Development and operation of a perfusion bioreactor for the cultivation of mammalian cells inside a sponge-like ceramic matrix [Elektronische Ressource] / vorgelegt von Vicky Goralczyk

Dipl.-Ing. Vicky Goralczyk

Sujets

• Biologie

Informations

• Publié par: technische_universitat_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 20 Mo

Extrait

Development and operation of a
perfusion bioreactor for the cultivation of
mammalian cells inside a sponge-like ceramic
matrix
vorgelegt von
Dipl.-Ing.
Vicky Goralczyk
aus Berlin
Von der Fakultät III - Prozesswissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktor der Ingenieurwissenschaften
Dr.-Ing.
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof.Dr.rer.nat.Lothar W.Kroh
Gutachter: Prof.Dr.-Ing.habil.Rudibert King
Gutachter: Prof.Dr.-Ing.Udo Reichl
Gutachter: Prof.Dr.rer.nat.Helmut Schubert
Tag der wissenschaftlichen Aussprache: 13.Juli 2010
Berlin 2010
D83i Contents
Contents
1 Abstract 1
2 Introduction 5
2.1 State of the art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1.1 Ceramics for cell cultivation . . . . . . . . . . . . . . . . . . . . . 6
2.1.2 Cell cultivation modes . . . . . . . . . . . . . . . . . . . . . . . . 10
2.1.3 Inoculation modes . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.1.4 Perfusion dynamic . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.2 Goals of this thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3 Materials and methods 19
3.1 Preparation of alumina ceramics for cell culture . . . . . . . . . . . . . . 19
3.2 Reactor device for cell cultivation on alumina foams . . . . . . . . . . . . 20
3.2.1 Tubular design for series connection of ceramics . . . . . . . . . . 21
3.2.2 Revolver design for parallel of ceramics . . . . . . . . . 22
3.2.3 Small block design for single foam analysis . . . . . . . . . . . . . 24
3.3 Inoculation procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.3.1 Static inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.3.2 Dynamic inoculation by stirring or agitation . . . . . . . . . . . . 26
3.3.3 ino by convectional forces . . . . . . . . . . . . . 26
3.4 Cultivation of cells on ceramics . . . . . . . . . . . . . . . . . . . . . . . 27
3.4.1 Perfusion cultivation inside the reactor device . . . . . . . . . . . 27
3.4.2 Static cultivation of cells outside the reactor device . . . . . . . . 29
3.4.3 Cultivation of cells by medium convection outside the reactor device 29
3.5 Cell lines and origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.6 Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.6.1 MicroscopicevaluationofcellvitalitybydyeingaccordingtoFDA/EB
protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Contents ii
3.6.2 Microscopic evaluation of cell distribution by dyeing according to
hematoxylin/eosin protocol . . . . . . . . . . . . . . . . . . . . . 32
3.6.3 Scanning electron microscopy of ceramic surface and cells on ce-
ramics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.6.4 Metabolic evaluation of glucose consumption and lactate formation 33
3.6.5 Reduction of resazurin . . . . . . . . . . . . . . . . . . . . . . . . 34
3.6.6 Carrier hot gas extraction . . . . . . . . . . . . . . . . . . . . . . 35
4 Reactor characterization 37
4.1 Ceramics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.1.1 Porosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.1.2 Flow resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.2 Characterization of flow inside the reactor . . . . . . . . . . . . . . . . . 39
5 AnalysisofCHO-K1 byresazurinassayandcarboncontentdetermination. 43
5.1 Reduction of resazurin by CHO-K1 . . . . . . . . . . . . . . . . . . . . . 43
5.1.1 Determination of rate of reduction . . . . . . . . . . . . . . . . . 43
5.1.2 Modeling resazurin reduction . . . . . . . . . . . . . . . . . . . . 46
5.1.3 Adaptationoftheresazurinreductionmodeltodescribebioreactor
performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.2 Carbon content of cells cultivated on ceramics . . . . . . . . . . . . . . . 54
5.2.1 Carbon content of pure cells . . . . . . . . . . . . . . . . . . . . . 54
5.2.2 Carbon content of cells on foams . . . . . . . . . . . . . . . . . . 55
6 Influence of mode of inoculation on cellular growth and distribution 59
6.1 Static inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
6.1.1 Static inoculation on foams in culture plates . . . . . . . . . . . . 59
6.1.2 Static ino into the tubular reactor . . . . . . . . . . . . . 60
6.1.3 Static inoculation into revolver reactors . . . . . . . . . . . . . . . 61
6.1.4 Static ino in ceramics with flow channels . . . . . . . . . . 65
6.1.5 Reproducible cultivation following static inoculation into revolver
reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
6.2 Dynamic inoculation by agitation . . . . . . . . . . . . . . . . . . . . . . 72
6.3 Oscillatory perfusion inoculation . . . . . . . . . . . . . . . . . . . . . . . 75
6.3.1 Influence of module orientation, flow velocity, initial cell count on
cell distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75iii Contents
6.3.2 Introduction of more porous ceramics and augmentation of flow
velocity during cultivation . . . . . . . . . . . . . . . . . . . . . . 76
6.3.3 Reduction of foam volume by reducing cylinder height . . . . . . 81
7 Reproducibility of the chosen operation methods 85
7.1 Standard foams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
7.2 Foams with larger pores . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
8 Long-term cultivation 97
9 Applicability of the reactor system for other cell types 103
9.1 Human lung carcinoma cells A549 . . . . . . . . . . . . . . . . . . . . . . 103
9.2 primary fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . 105
9.3 Madin-Darby canine kidney cells (MDCK) . . . . . . . . . . . . . . . . . 106
10 Conclusion 111
Appendix 117
Literature 130v Abbreviatons and Symbols
Abbreviations
Al O aluminium oxide2 3
CO carbon dioxide2
CHGE carrier hot gas extraction
DNA desoxyribonucleic acid
EB ethidium bromide
ECM extracellular matrix
FBS fetal bovine serum
FDA fluorescein diacetate
HA hydroxy-apatite
IFS Interdisciplinary Research Priority Program
PBS++ phosphate buffered saline containing magnesium and calcium
PFR plug flow reactor
ppi pores per inch
SD standard deviation
SEM scanning electron microscopy
STR stirred tank reactor
TCP tri-calcium phosphate
TU Berlin Technische Universität Berlinvii Abbreviatons and Symbols
Symbols
p pressure drop [Pa]
_V volumetric flow [ml/min]
coefficient of viscosity [mPas]
porosity
density [g/ml]
standard deviation
normalized time
2A area [m ]
Bo Bodenstein number
c concentration [g/l]
2D axial coefficient of dispersion [m /s]ax
E extinction
F normalized extinction
H height [m]
2K Darcy’s constant [m ]d
L length [m]
m mass [g]
n quantityAbbreviatons and Symbols viii
p percentage
2R coefficient of determination
t time [s]
V volume [ml]
x normalized concentration